Eric W. Mills

The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel

TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe3+-bound transferrin receptors, or after lysosomal degradation of ferritin–iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe2+ transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe2+ transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe2+ permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe2+ at varying degrees, which correlate well with the disease severity. A comparison of TRPML1-/- ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe2+ levels, an increase in intralysosomal Fe2+ levels and an accumulation of lipofuscin-like molecules in TRPML1-/- cells. We propose that TRPML1 mediates a mechanism by which Fe2+ is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.

An Essential Mesenchymal Function for miR-143/145 in Intestinal Epithelial Regeneration

Downregulation of the miR-143/145 microRNA (miRNA) cluster has been repeatedly reported in colon cancer and other epithelial tumors. In addition, overexpression of these miRNAs inhibits tumorigenesis, leading to broad consensus that they function as cell-autonomous epithelial tumor suppressors. We generated mice with deletion of miR-143/145 to investigate the functions of these miRNAs in intestinal physiology and disease in vivo. Although intestinal development proceeded normally in the absence of these miRNAs, epithelial regeneration after injury was dramatically impaired. Surprisingly, we found that miR-143/145 are expressed and function exclusively within the mesenchymal compartment of intestine. Defective epithelial regeneration in miR-143/145-deficient mice resulted from the dysfunction of smooth muscle and myofibroblasts and was associated with derepression of the miR-143 target Igfbp5, which impaired IGF signaling after epithelial injury. These results provide important insights into the regulation of epithelial wound healing and argue against a cell-autonomous tumor suppressor role for miR-143/145 in colon cancer.

Activating Mutations of the TRPML1 Channel Revealed by Proline-scanning Mutagenesis

The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca2+-dependent, constitutive Ca2+ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca2+ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM.

A Bicistronic MAVS Transcript Highlights a Class of Truncated Variants in Antiviral Immunity

Bacterial and viral mRNAs are often polycistronic. Akin to alternative splicing, alternative translation of polycistronic messages is a mechanism to generate protein diversity and regulate gene function. Although a few examples exist, the use of polycistronic messages in mammalian cells is not widely appreciated. Here we report an example of alternative translation as a means of regulating innate immune signaling. MAVS, a regulator of antiviral innate immunity, is expressed from a bicistronic mRNA encoding a second protein, miniMAVS. This truncated variant interferes with interferon production induced by full-length MAVS, whereas both proteins positively regulate cell death. To identify other polycistronic messages, we carried out genome-wide ribosomal profiling and identified a class of antiviral truncated variants. This study therefore reveals the existence of a functionally important bicistronic antiviral mRNA and suggests a widespread role for polycistronic mRNAs in the innate immune system.

Ribosomopathies: There’s strength in numbers

Molecular mechanisms behind ribosomopathies Ribosomopathies are t issuespecific disorders that result from mutations in ribosomal proteins or ribosome biogenesis factors. Such disorders include Diamond-Blackfan anemia, isolated congenital asplenia, and Treacher Collins syndrome. Mills and Green review the underlying mechanisms of tissue-specific defects in these and related disorders. Because ribosomes are central to all cellular life, it is puzzling why mutations in components of the ribosome disproportionately affect certain tissues. The authors suggest that ribosome homeostasis is an overarching and simplifying principle that governs the sensitivity of specific cells and tissue types to mutation in components of the translational machinery. Science, this issue p. eaan2755 BACKGROUND Ribosomopathies are a heterogeneous group of human disorders that are in some cases known, and in other cases suspected, to result from ribosome dysfunction. This group broadly comprises two categories: (i) disorders caused by single-copy mutations in specific ribosomal proteins, and (ii) disorders associated with defects in ribosome biogenesis factors. The phenotypic patterns among different ribosomopathies in both categories are divergent but do tend to share some overlapping features. These include effects on bone marrow–derived cell lineages and skeletal tissues. These common tissue specificities of the different ribosomopathies are challenging to reconcile with the ubiquitous requirement for ribosomes in all cells. ADVANCES Several models have been advanced to explain how the dysfunction of the protein synthesis machinery is so variably expressed at the phenotypic level. An increasing number of studies in models of distinct ribosomopathies have revealed that “ribosomal stress” signals converge on the p53 signaling pathway in affected cells and tissues. In these models, a key consequence of ribosome dysfunction is cell- and tissue type–restricted activation of p53-dependent cell cycle arrest and apoptosis. However, the specific translational events upstream of p53 activation that lead to some cells being affected, with others being spared, are unknown. We review evidence relating to two hypotheses that have been proposed to explain such tissue-specific effects of ribosome dysfunction. One hypothesis is that ribosome dysfunction (or deficiency) can affect global and messenger RNA (mRNA)–specific translational control, and that certain specific cells or tissues may be more vulnerable to ribosome dysfunction. A critical feature of this view is that mRNAs are variably dependent on cellular ribosome concentration, with more poorly initiated mRNAs being typically more sensitive to perturbations in ribosome concentration or function. Several recent studies suggest that the sensitivities of certain tissues to ribosomopathies, including reticuloctyes and platelets, may be related to differences in core processes of translation in these cells related to ribosome recycling and rescue. Perturbations in these processes will have a great impact on ribosome homeostasis and thus on broad aspects of gene expression. Related studies in the brain have revealed disease phenotypes in genetic backgrounds with deficiencies in ribosome rescue and in a specific neuronal transfer RNA. Together, these molecular insights provide a new perspective on ribosomopathies and their tissue specificities, while also raising a number of important questions to pursue. The other hypothesis is that ribosomes from different tissues may have different compositions of core or more loosely associated proteins and posttranslational modifications, and that this heterogeneity could be critical to the translation of specific mRNAs. This is referred to as the “specialized” ribosome hypothesis. We argue that while such heterogeneity in ribosome composition likely exists in different tissues, such complex explanations may not be needed to explain the differences in gene expression that result from losses of specific ribosomal proteins. It is simpler to hypothesize that differences in mRNA-specific rates of initiation and changes in ribosome concentration can adequately explain much (if not all) of the diversity of gene expression changes in different tissues as a result of ribosomal mutations. OUTLOOK A cohesive mechanistic model connecting dysfunction of the ribosome to the specific phenotypic consequences observed in ribosomopathies remains a challenging goal. For example, it is inherently difficult to assess the function of particular ribosomes in a cell, and thus to differentiate among various models to explain the impacts of ribosome deficiencies on gene expression. Further biochemical analyses of the fundamental processes underlying the cellular response to protein synthesis dysfunction, refinements in cellular and animal models of ribosomopathies, and greater dialogue between clinical and basic scientists will all be important to extend our current understanding. Ribosome concentration drives mRNA-specific effects on translation. The translation rate varies as a function of cellular ribosome concentration and mRNA-specific initiation rates (heat map, left); a scatterplot model of ribosome footprinting data (right) shows how ribosome deficiency would be predicted to have varying effects on different mRNAs. The black, gray, and orange boxes define groups of mRNAs of similar initiation efficiencies as they respond to changes in ribosome concentration (as represented by the same colors at right). Ribosomopathies are a group of human disorders most commonly caused by ribosomal protein haploinsufficiency or defects in ribosome biogenesis. These conditions manifest themselves as physiological defects in specific cell and tissue types. We review current molecular models to explain ribosomopathies and attempt to reconcile the tissue specificity of these disorders with the ubiquitous requirement for ribosomes in all cells. Ribosomopathies as a group are diverse in their origins and clinical manifestations; we use the well-described Diamond-Blackfan anemia (DBA) as a specific example to highlight some common features. We discuss ribosome homeostasis as an overarching principle that governs the sensitivity of specific cells and tissue types to ribosomal protein mutations. Mathematical models and experimental insights rationalize how even subtle shifts in the availability of ribosomes, such as those created by ribosome haploinsufficiency, can drive messenger RNA–specific effects on protein expression. We discuss recently identified roles played by ribosome rescue and recycling factors in regulating ribosome homeostasis.

Dependence on a retinophilin/myosin complex for stability of PKC and INAD and termination of phototransduction.

Normal termination of signaling is essential to reset signaling cascades, especially those such as phototransduction that are turned on and off with great rapidity. Genetic approaches in Drosophila led to the identification of several proteins required for termination, including protein kinase C (PKC), NINAC (neither inactivation nor afterpotential C) p174, which consists of fused protein kinase and myosin domains, and a PDZ (postsynaptic density-95/Discs Large/zona occludens-1) scaffold protein, INAD (inactivation no afterpotential D). Here, we describe a mutation affecting a poorly characterized but evolutionarily conserved protein, Retinophilin (Retin), which is expressed primarily in the phototransducing compartment of photoreceptor cells, the rhabdomeres. Retin and NINAC formed a complex and were mutually dependent on each other for expression. Loss of retin resulted in an age-dependent impairment in termination of phototransduction. Mutations that affect termination of the photoresponse typically lead to a reduction in levels of the major rhodopsin (Rh1) to attenuate signaling. Consistent with the slower termination in retin1, the mutant photoreceptor cells exhibited increased endocytosis of Rh1 and a decline in Rh1 protein. The slower termination in retin1 was a consequence of a cascade of defects, which began with the reduction in NINAC p174 levels. The diminished p174 concentration caused a decrease in INAD. Because PKC requires interaction with INAD for protein stability, this leads to reduction in PKC levels. The decline in PKC was age dependent and paralleled the onset of the termination phenotype in retin1 mutant flies. We conclude that the slower termination of the photoresponse in retin1 resulted from a requirement for the Retin/NINAC complex for stability of INAD and PKC.

Slowed decay of mRNAs enhances platelet specific translation

Platelets are anucleate cytoplasmic fragments that lack genomic DNA, but continue to synthesize protein using a pool of messenger RNAs (mRNAs), ribosomes, and regulatory small RNAs inherited from the precursor megakaryocyte (MK). The regulatory processes that shape the platelet transcriptome and the full scope of platelet translation have remained elusive. Using RNA sequencing (RNA-Seq) and ribosome profiling of primary human platelets, we show the platelet transcriptome encompasses a subset of transcripts detected by RNA-Seq analysis of in vitro-derived MK cells and that these platelet-enriched transcripts are broadly occupied by ribosomes. We use RNA-Seq of synchronized populations of in vitro-derived platelet-like particles to show that mRNA decay strongly shapes the nascent platelet transcriptome. Our data suggest that the decay of platelet mRNAs is slowed by the natural loss of the mRNA surveillance and ribosome rescue factor Pelota.

Case of Secondary Tics Associated With Olanzapine in an Adult

Atypical antipsychotic medications, such as risperidone, aripiprazole, and olanzapine, have utility in treating motor tics, particularly in Tourette syndrome. In rare cases, atypical antipsychotic medications have been associated with adult-onset motor tics. Such adverse drug reactions have been documented in response to quetiapine, aripiprazole, and amisulpride. Here, we report, to our knowledge, the first case of adult-onset motor tics related to olanzapine administration.